Micro-pit preparation for single molecule detection chip substrate and single molecule detection chip

Abstract

This invention belongs to the field of materials preparation technology, specifically relating to the fabrication of micropits for single-molecule detection chip substrates and the single-molecule detection chip itself. The method provided by this invention mainly includes the following steps: first, heavy ions are accelerated using an accelerator; then, the accelerated heavy ions bombard the substrate. After the heavy ions damage the substrate, an etchant with added corrosion inhibitors is used to etch the substrate, obtaining micropits of specific diameter and depth. The etched sample is then cleaned and dried. This method of fabricating micropits by accelerating and bombarding the substrate with heavy ions overcomes the limitations of traditional photolithography. It directly defines the micropit pattern through heavy ion bombardment, eliminating the need for photomasks. This not only reduces material costs but also avoids production interruptions and additional costs caused by photomask damage. Furthermore, the processing is relatively simple, eliminating the need for multiple complex photolithography and etching steps, greatly improving production efficiency, shortening the production cycle, and reducing production costs.

Description

Technical Field

[0001] This invention belongs to the field of materials preparation technology, specifically relating to the preparation of micropits for single-molecule detection chip substrates and single-molecule detection chips. Background Technology

[0002] With the continuous advancement of science and technology, a variety of novel detection technologies with high sensitivity, strong specificity, and rapid diagnostic capabilities are rapidly developing and are increasingly being applied to the detection of animal diseases. Among them, single-molecule detection technology can study the structure and function of biomolecules at the single-molecule level and can be used to detect low-abundance biomarkers in complex samples. It has enormous application potential in single-molecule level imaging and tracking of interactions between low-concentration proteins, nucleic acids, and other biologically related molecules, ultrasensitive quantitative analysis, and precision medicine, and has been widely used in early disease diagnosis, exploration of potential diseases, live-cell single-virus tracing, and DNA sequencing.

[0003] Digital ELISA, as an emerging single-molecule detection technology, achieves single-molecule-level detection by dispersing single molecules into high-throughput microreactors for independent reactions and by detecting and statistically analyzing the signals from each microreactor. For example, the "Digital ELISA" technology developed by David Walt and David Duffy's team in 2010 uses labeled β-galactosidase to catalyze the substrate to generate a fluorescent signal, replacing the PCR signal amplification process in digital PCR. This allows single-molecule proteins labeled with β-galactosidase to be dispersed into high-throughput microreactors for independent reactions, thus achieving extremely high detection sensitivity. However, digital ELISA technology still faces many challenges in practical applications. Microreactor-based digital ELISA suffers from problems such as high chip fabrication difficulty and cost, and low production efficiency. Currently, the main method for fabricating microreactor detection chips is photolithography. Photolithography requires high-precision masks, complex photolithography equipment, and cumbersome experimental operations. These factors not only increase the cost of chip fabrication but also reduce its efficiency. Significant difficulties remain both technically and commercially.

[0004] Therefore, it is crucial to develop more stable fabrication methods for single-molecule detection micro-pits and novel single-molecule detection chips to improve chip fabrication efficiency, reduce chip fabrication costs, and enhance their commercial value. Summary of the Invention

[0005] To address the aforementioned challenges, this invention provides a method for fabricating micropits on a substrate for a single-molecule detection chip. By using a high-energy accelerator to accelerate heavy ion bombardment of the substrate, the limitations of traditional photolithography are overcome, significantly reducing the cost of micropit fabrication and effectively solving the problems and defects existing in the prior art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for fabricating micropits for a single-molecule detection chip substrate includes the following steps:

[0008] S1. Irradiation: Heavy ions are accelerated using an accelerator to obtain a heavy ion beam, and then the heavy ion beam is used to bombard the substrate to generate nuclear tracks, thereby obtaining a damaged substrate.

[0009] S2, Etching: Etching the damaged substrate obtained in step S1 with an etchant to obtain a substrate containing micropits;

[0010] S3. Cleaning and drying: Clean the substrate containing micropits obtained in step S2 with cleaning solution, and dry it in a vacuum drying oven to obtain the substrate for single-molecule detection chip.

[0011] Preferably, in step S1, the heavy ions are accelerated to an energy of 10~1200MeV using an accelerator, and the heavy ions include Br ions and Ar ions.

[0012] Preferably, the substrate in step S1 is one of glass, silicon wafer and polymer film; the thickness of the target substrate is 0.002~6mm.

[0013] Preferably, the heavy ion beam described in step S1 is scanned into a rectangular plane using a beam scanner.

[0014] Preferably, in step S1, during the bombardment of the substrate with accelerated heavy ions, the substrate is bombarded with heavy ions in the atmosphere, and a titanium film is used for vacuum isolation. After bombarding the substrate, the density of the nuclear track is 1 × 10⁻⁶ per square centimeter. 4 ~1×10 9 ions.

[0015] In actual fabrication, the structure, thickness, and mechanical properties of the substrate all affect the formation and density of micropits. Therefore, the acceleration energy differs when heavy ions are accelerated before bombarding the substrate during irradiation: the thickness of the glass slide is generally 0.002~1mm, and the accelerated heavy ion energy is 100~1000MeV, with a nuclear track density of 1×10⁻⁶. 4 ~5×10 7 ions / cm 2 The length of the nuclear tracks ranges from 0.1 to 50 micrometers; the thickness of the polycarbonate film ranges from 0.002 to 5 mm; the heavy ion energy after acceleration is 50 to 1000 MeV; and the density of the nuclear tracks is 1 × 10⁻⁶. 4 ~1×10 9 ions / cm 2The length of the nuclear tracks ranges from 0.1 to 50 micrometers; the thickness of the polyester film ranges from 0.002 to 3 mm; the energy of the accelerated heavy ions is 50 to 600 MeV; and the density of the nuclear tracks is 1 × 10⁻⁶. 4 ~5×10 7 ions / cm 2 The length of the nuclear tracks ranges from 0.1 to 50 micrometers; the thickness of the polyimide film ranges from 0.002 to 5 mm; the heavy ion energy after acceleration is 10 to 800 MeV; and the density of the nuclear tracks on the polyimide film is 1 × 10⁻⁶. 4 ~1×10 9 ions / cm 2 The length of the nuclear track ranges from 0.1 to 50 micrometers.

[0016] Preferably, the etchant in step S2 is one of hydrogen fluoride solution, sodium hydroxide solution, potassium hydroxide solution, and fluorosilicic acid solution.

[0017] Preferably, the etchant in step S2 further includes a corrosion inhibitor, which is selected from one or more of methionine, histidine, molybdate, and tungstate.

[0018] Preferably, the etching temperature in step S2 is 25~80℃ and the etching time is 90s~180min; the diameter of the micropits on the substrate obtained in step S2 is 0.3~45μm and the depth of the micropits is 0.2~30μm.

[0019] Different substrates directly affect the selection of etching solution, etching temperature, and etching time. By adjusting the etching temperature and etching time, micropits with adjustable diameter and depth can be obtained. Therefore, in the actual micropit fabrication process, it is necessary to precisely select etching conditions: for glass slides, chemical etching is performed on irradiated glass slides using a 0.1~10 mol / L HF solution at a temperature of 20~60℃ for 90~600 s; for polycarbonate films, chemical etching is performed on irradiated films using a 0.1~15 mol / L NaOH solution at a temperature of 50~80℃ for 1~120 min; for polyester films, chemical etching is performed on irradiated films using a 0.1~15 mol / L NaOH solution at a temperature of 50~80℃ for 1~180 min; and for polyimide films, chemical etching is performed on irradiated films using a 0.1~10 mol / L NaOH solution at a temperature of 30~80℃ for 1~150 min.

[0020] However, due to the surface damage, structural changes, and enhanced chemical activity of the substrate after heavy ion bombardment, the damaged substrate is more susceptible to corrosion. If etching time and other conditions are not strictly controlled, over-corrosion can easily occur, affecting the substrate quality. Therefore, to reduce the difficulty of process control, the etchant in step S2 of this invention also includes a corrosion inhibitor, which is selected from one or more of methionine, histidine, molybdate, and tungstate.

[0021] This invention incorporates a corrosion inhibitor during the etching process to prevent excessive erosion of the substrate due to untimely monitoring or process control errors. Furthermore, by controlling the etching rate, the etching process can be monitored in real time, allowing for timely adjustments to bombardment angles and heavy ion energy. This enables the fabrication of micropits in various shapes, including cylindrical, conical, symmetrical biconical, and asymmetrical biconical. The corrosion inhibitor prevents excessive corrosion caused by high-speed etching, thus avoiding damage to the designed shape. In particular, the biconical micropits, through their unique geometry, perfectly combine optics, fluid dynamics, and surface chemistry, achieving the goal of long-term, high signal-to-noise ratio, real-time observation of single-molecule behavior, thereby driving the birth and development of disruptive technologies such as single-molecule sequencing.

[0022] In the process of researching corrosion inhibitors, this invention discovered that for acidic etching solutions, methionine or histidine can be selected as a corrosion inhibitor, which has a good corrosion inhibition effect and can protect the substrate; for alkaline etching solutions, molybdate or tungstate can be selected as a corrosion inhibitor, which can protect the substrate and avoid excessive corrosion.

[0023] The present invention also provides a substrate for a single-molecule detection chip prepared by the above preparation method.

[0024] The present invention also provides a single-molecule detection chip comprising the substrate.

[0025] Compared with existing technologies, this invention achieves the fabrication of micropits by accelerating the bombardment of the substrate with heavy ions. This method breaks through the limitations of traditional photolithography technology and has significant advantages and beneficial effects:

[0026] (1) Traditional photolithography requires the use of high-precision photomasks to define the pattern of micro-pits. The production cost of photomasks is very high and they are easily damaged. However, this invention defines the pattern of micro-pits by heavy ion bombardment, which eliminates the need for photomasks. This not only reduces material costs but also avoids production interruptions and additional costs caused by photomask damage.

[0027] (2) The present invention uses heavy ion bombardment to not only control the size of the micro-pit, but also to achieve more complex shape control. By adjusting the energy and bombardment angle of the heavy ions, micro-pits with different shapes can be prepared, such as circular, square, elliptical, etc., and even micro-pits with special structures, such as conical micro-pits, stepped micro-pits, etc., providing more possibilities for the application of micro-pits.

[0028] (3) The processing of the present invention is relatively simple, without the need for multiple complex photolithography and etching steps. The micro-pits can be prepared in one heavy ion bombardment and etching process. This greatly improves production efficiency, shortens the production cycle, and reduces production costs. Attached Figure Description

[0029] Figure 1 Schematic diagrams of different shapes of micropits made from glass sheets as substrates;

[0030] Figure 2 This is a schematic diagram of the micro-pits on the glass slide in Example 1;

[0031] Figure 3 This is a schematic diagram of the micro-pits in the polyester film (PET) in Example 6;

[0032] Figure 4 This is a schematic diagram of the micro-pits in the polycarbonate (PC) film in Example 11;

[0033] Figure 5 This is a schematic diagram of the micro-pits in the polyimide (PI) film in Example 16. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are only for better explanation of the present invention and do not limit the technical solutions of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The reagents and equipment used in this invention are all conventional reagents and equipment in the art.

[0035] Example 1: Method for fabricating micropits for single-molecule detection chip substrate

[0036] The specific process for fabricating micropits using a glass slide as a substrate is as follows:

[0037] S1. Irradiation: Br ions are accelerated to an energy of 140 MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a glass slide with a thickness of 0.02 mm to generate nuclear tracks on the glass slide and obtain a damaged glass slide.

[0038] S2, Etching: Using a 0.35 mol / L HF solution, chemical etching is performed on the damaged glass sheet obtained in step S1 at a temperature of 25°C for 90 seconds to obtain a glass sheet containing micropits.

[0039] S3. Cleaning and drying: The glass slide containing micropits obtained in step S2 is cleaned repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a glass slide with micropits. Figure 2 By cutting glass slides with micropits, single-molecule detection chips can be fabricated.

[0040] Example 2: Method for fabricating micropits for single-molecule detection chip substrate

[0041] The specific process for fabricating micropits using a glass slide as a substrate is as follows:

[0042] S1. Irradiation: Br ions are accelerated to an energy of 280 MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a glass slide with a thickness of 0.1 mm to produce nuclear tracks on the glass slide and obtain a damaged glass slide.

[0043] S2. Etching: Using a 1 mol / L HF solution, chemical etching is performed on the irradiated glass slide at a temperature of 25°C for 200 seconds to obtain a glass slide containing micropits.

[0044] S3. Cleaning and Drying: The glass slide containing micropits obtained in step S2 is cleaned multiple times with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a glass slide with micropits. The glass slide with micropits is then cut to make a chip for single-molecule detection.

[0045] Example 3: Method for fabricating micropits for single-molecule detection chip substrate

[0046] The specific process for fabricating micropits using a glass slide as a substrate is as follows:

[0047] S1. Irradiation: Br ions are accelerated to an energy of 560 MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a glass slide with a thickness of 0.35 mm to generate nuclear tracks on the glass slide and obtain a damaged glass slide.

[0048] S2. Etching: Using a 4 mol / L HF solution, chemical etching is performed on the irradiated glass slide at a temperature of 50°C for 360 seconds to obtain a glass slide containing micropits.

[0049] S3. Cleaning and Drying: The glass slide containing micropits obtained in step S2 is cleaned multiple times with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a glass slide with micropits. The glass slide with micropits is then cut to make a chip for single-molecule detection.

[0050] Example 4: Method for fabricating micropits for single-molecule detection chip substrate

[0051] The specific process for fabricating micropits using a glass slide as a substrate is as follows:

[0052] S1. Irradiation: Br ions are accelerated to an energy of 980 MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a glass slide with a thickness of 1 mm to produce nuclear tracks on the glass slide, thus obtaining a damaged glass slide.

[0053] S2. Etching: Using a 10 mol / L HF solution, chemical etching is performed on the irradiated glass slide at a temperature of 50°C for 500 seconds to obtain a glass slide containing micropits.

[0054] S3. Cleaning and Drying: The glass slide containing micropits obtained in step S2 is cleaned multiple times with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a glass slide with micropits. The glass slide with micropits is then cut to make a chip for single-molecule detection.

[0055] Example 5: Method for fabricating micropits for single-molecule detection chip substrate

[0056] The specific process for fabricating micropits using a glass slide as a substrate is as follows:

[0057] S1. Irradiation: Br ions are accelerated to an energy of 980 MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a glass slide with a thickness of 1 mm to produce nuclear tracks on the glass slide, thus obtaining a damaged glass slide.

[0058] S2. Etching: Using a 10 mol / L HF solution, a certain weight of methionine is added to the solution until the final concentration of methionine is 0.7 g / L. The irradiated glass slide is chemically etched at a temperature of 50°C for 600 s to obtain a glass slide containing micropits.

[0059] S3. Cleaning and Drying: The glass slide containing micropits obtained in step S2 is cleaned multiple times with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a glass slide with micropits. The glass slide with micropits is then cut to make a chip for single-molecule detection.

[0060] Example 6: Method for fabricating micropits for single-molecule detection chip substrate

[0061] The micropits were fabricated using polyester film as a substrate, and the specific process is as follows:

[0062] S1. Irradiation: Ar ions are accelerated to an energy of 70 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, which is then used to irradiate a polyester film with a thickness of 0.01 mm to generate nuclear tracks on the polyester film, thus obtaining a damaged polyester film.

[0063] S2. Etching: Using a 2 mol / L NaOH solution, the irradiated polyester film was chemically etched at 60°C for 15 minutes to obtain a polyester film containing micropits.

[0064] S3. Cleaning and drying: The polyester film containing micro-pits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyester film with micro-pits. Figure 3 By cutting a polyester film with micropits, a chip for single-molecule detection can be made.

[0065] Example 7: Method for fabricating micropits for single-molecule detection chip substrate

[0066] The micropits were fabricated using polyester film as a substrate, and the specific process is as follows:

[0067] S1. Irradiation: Ar ions are accelerated to an energy of 210 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, which is then used to irradiate a polyester film with a thickness of 0.1 mm to generate nuclear tracks on the polyester film, thus obtaining a damaged polyester film.

[0068] S2. Etching: Using a 5 mol / L NaOH solution, the irradiated polyester film was chemically etched at a temperature of 60℃ for 45 min to obtain a polyester film containing micropits.

[0069] S3. Cleaning and Drying: The polyester film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyester film with micropits. The polyester film with micropits is then cut to make a chip for single-molecule detection.

[0070] Example 8: Method for fabricating micropits for single-molecule detection chip substrate

[0071] The micropits were fabricated using polyester film as a substrate, and the specific process is as follows:

[0072] S1. Irradiation: Accelerate Ar ions to an energy of 490 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, and then irradiate a polyester film with a thickness of 1 mm to generate nuclear tracks on the polyester film, thus obtaining a damaged polyester film.

[0073] S2. Etching: Using a 10 mol / L NaOH solution, the irradiated polyester film was chemically etched at 80°C for 90 min to obtain a polyester film containing micropits.

[0074] S3. Cleaning and Drying: The polyester film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyester film with micropits. The polyester film with micropits is then cut to make a chip for single-molecule detection.

[0075] Example 9: Method for fabricating micropits for single-molecule detection chip substrate

[0076] The micropits were fabricated using polyester film as a substrate, and the specific process is as follows:

[0077] S1. Irradiation: Accelerate Ar ions to an energy of 560 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, and then irradiate a polyester film with a thickness of 2 mm to generate nuclear tracks on the polyester film, thus obtaining a damaged polyester film.

[0078] S2. Etching: Using a 15 mol / L NaOH solution, the irradiated polyester film was chemically etched at 80°C for 180 min to obtain a polyester film containing micropits.

[0079] S3. Cleaning and Drying: The polyester film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyester film with micropits. The polyester film with micropits is then cut to make a chip for single-molecule detection.

[0080] Example 10: Method for fabricating micropits for single-molecule detection chip substrate

[0081] The micropits were fabricated using polyester film as a substrate, and the specific process is as follows:

[0082] S1. Irradiation: Accelerate Ar ions to an energy of 560 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, and then irradiate a polyester film with a thickness of 2 mm to generate nuclear tracks on the polyester film, thus obtaining a damaged polyester film.

[0083] S2. Etching: Using a 15 mol / L NaOH solution, a certain amount of sodium molybdate is added to the solution until the final concentration of sodium molybdate is 0.9 g / L. The irradiated polyester film is chemically etched at a temperature of 80℃ for 200 min to obtain a polyester film containing micropits.

[0084] S3. Cleaning and Drying: The polyester film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyester film with micropits. The polyester film with micropits is then cut to make a chip for single-molecule detection.

[0085] Example 11: Method for fabricating micropits for single-molecule detection chip substrate

[0086] The micropits were fabricated using polycarbonate film as a substrate, and the specific process is as follows:

[0087] S1. Irradiation: Ar ions are accelerated to an energy of 70 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, which is then used to irradiate a polycarbonate film with a thickness of 0.01 mm to generate nuclear tracks on the polycarbonate film, thus obtaining a damaged polycarbonate film.

[0088] S2. Etching: Using a 1 mol / L NaOH solution, the irradiated polycarbonate film was chemically etched at 50°C for 5 minutes to obtain a polycarbonate film containing micropits.

[0089] S3. Cleaning and drying: The polycarbonate film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polycarbonate film with micropits. Figure 4 By cutting polycarbonate films with micropits, single-molecule detection chips can be fabricated.

[0090] Example 12: Method for fabricating micropits for single-molecule detection chip substrate

[0091] The micropits were fabricated using polycarbonate film as a substrate, and the specific process is as follows:

[0092] S1. Irradiation: Ar ions are accelerated to an energy of 210 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, which is then used to irradiate a 0.1 mm thick polycarbonate film to generate nuclear tracks on the polycarbonate film and obtain a damaged polycarbonate film.

[0093] S2. Etching: Using a 5 mol / L NaOH solution, the irradiated polycarbonate film was chemically etched at 50°C for 20 min to obtain a polycarbonate film containing micropits.

[0094] S3. Cleaning and Drying: The polycarbonate film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polycarbonate film with micropits. The polycarbonate film with micropits can be cut to make a chip for single-molecule detection.

[0095] Example 13: Method for fabricating micropits for single-molecule detection chip substrate

[0096] The micropits were fabricated using polycarbonate film as a substrate, and the specific process is as follows:

[0097] S1. Irradiation: Ar ions are accelerated to an energy of 630 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, which is then used to irradiate a 2 mm thick polycarbonate film to generate nuclear tracks on the polycarbonate film, thus obtaining a damaged polycarbonate film.

[0098] S2. Etching: Using a 10 mol / L NaOH solution, the irradiated polycarbonate film was chemically etched at 70°C for 60 min to obtain a polycarbonate film containing micropits.

[0099] S3. Cleaning and Drying: The polycarbonate film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polycarbonate film with micropits. The polycarbonate film with micropits can be cut to make a chip for single-molecule detection.

[0100] Example 14: Method for fabricating micropits for single-molecule detection chip substrate

[0101] The method for preparing micropits using polycarbonate film as a substrate is as follows:

[0102] S1. Irradiation: Ar ions are accelerated to an energy of 980 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, which is then used to irradiate a 5 mm thick polycarbonate film to generate nuclear tracks on the polycarbonate film, thus obtaining a damaged polycarbonate film.

[0103] S2. Etching: Using a 15 mol / L NaOH solution, the irradiated polycarbonate film was chemically etched at 50°C for 100 min to obtain a polycarbonate film containing micropits.

[0104] S3. Cleaning and Drying: The polycarbonate film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polycarbonate film with micropits. The polycarbonate film with micropits can be cut to make a chip for single-molecule detection.

[0105] Example 15: Method for fabricating micropits for single-molecule detection chip substrate

[0106] The micropits were fabricated using polycarbonate film as a substrate, and the specific process is as follows:

[0107] S1. Irradiation: Ar ions are accelerated to an energy of 980 MeV using an accelerator to obtain a quasi-monoenergetic Ar ion beam, which is then used to irradiate a 5 mm thick polycarbonate film to generate nuclear tracks on the polycarbonate film, thus obtaining a damaged polycarbonate film.

[0108] S2. Etching: Using a 15 mol / L NaOH solution, a certain weight of sodium molybdate is added to the solution until the final concentration of sodium molybdate is 0.9 g / L. The irradiated polycarbonate film is chemically etched at a temperature of 50°C for 120 min to obtain a polycarbonate film containing micropits.

[0109] S3. Cleaning and Drying: The polycarbonate film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polycarbonate film with micropits. The polycarbonate film with micropits can be cut to make a chip for single-molecule detection.

[0110] Example 16: Method for fabricating micropits for single-molecule detection chip substrate

[0111] The micropits were fabricated using polyimide film as a substrate, and the specific process is as follows:

[0112] S1. Irradiation: Br ions are accelerated to an energy of 56MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a polyimide film with a thickness of 0.01mm to generate nuclear tracks on the polyimide film, thus obtaining a damaged polyimide film.

[0113] S2. Etching: The irradiated polyimide film was chemically etched using a 0.5 mol / L NaOH solution at 50°C for 20 min to obtain a polyimide film containing micropits.

[0114] S3. Cleaning and Drying: The polyimide film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyimide film with micropits. Figure 5 By cutting polyimide films with micropits, single-molecule detection chips can be fabricated.

[0115] Example 17: Method for fabricating micropits for single-molecule detection chip substrate

[0116] The micropits were fabricated using polyimide film as a substrate, and the specific process is as follows:

[0117] S1. Irradiation: Br ions are accelerated to an energy of 140 MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a 0.1 mm thick polyimide film to generate nuclear tracks on the polyimide film, thus obtaining a damaged polyimide film.

[0118] S2. Etching: Using a 1 mol / L NaOH solution, the irradiated polyimide film was chemically etched at 50°C for 40 min to obtain a polyimide film containing micropits.

[0119] S3. Cleaning and Drying: The polyimide film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyimide film with micropits. The polyimide film with micropits can be cut to make a chip for single-molecule detection.

[0120] Example 18: Method for fabricating micropits for single-molecule detection chip substrate

[0121] The micropits were fabricated using polyimide film as a substrate, and the specific process is as follows:

[0122] S1. Irradiation: Br ions are accelerated to an energy of 420MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a 1mm thick polyimide film to generate nuclear tracks on the polyimide film, thus obtaining a damaged polyimide film.

[0123] S2. Etching: The irradiated polyimide film was chemically etched using a 5 mol / L NaOH solution at 70°C for 100 min to obtain a polyimide film containing micropits.

[0124] S3. Cleaning and Drying: The polyimide film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyimide film with micropits. The polyimide film with micropits can be cut to make a chip for single-molecule detection.

[0125] Example 19: Method for fabricating micropits for single-molecule detection chip substrate

[0126] The method for preparing micropits using polyimide film as a substrate is as follows:

[0127] S1. Irradiation: Br ions are accelerated to an energy of 770 MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a 3 mm thick polyimide film to generate nuclear tracks on the polyimide film, thus obtaining a damaged polyimide film.

[0128] S2. Etching: Using a 10 mol / L NaOH solution, the irradiated polyimide film was chemically etched at 70°C for 120 min to obtain a polyimide film containing micropits.

[0129] S3. Cleaning and Drying: The polyimide film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyimide film with micropits. The polyimide film with micropits can be cut to make a chip for single-molecule detection.

[0130] Example 20: Method for fabricating micropits for single-molecule detection chip substrate

[0131] The micropits were fabricated using polyimide film as a substrate, and the specific process is as follows:

[0132] S1. Irradiation: Br ions are accelerated to an energy of 770 MeV using an accelerator to obtain a quasi-monoenergetic Br ion beam, which is then used to irradiate a 3 mm thick polyimide film to generate nuclear tracks on the polyimide film, thus obtaining a damaged polyimide film.

[0133] S2. Etching: Using a 10 mol / L NaOH solution, the irradiated polyimide film was chemically etched at 70°C for 120 min to obtain a polyimide film containing micropits.

[0134] S3. Cleaning and Drying: The polyimide film containing micropits obtained in step S2 is washed repeatedly with clean water and deionized water. After cleaning, it is dried in a vacuum drying oven to obtain a polyimide film with micropits. The polyimide film with micropits can be cut to make a chip for single-molecule detection.

[0135] Comparative Example 1: A method for preparing micropits

[0136] The method for preparing the micro-pits is similar to that in Example 5;

[0137] The difference from Example 5 is that in Comparative Example 1, methionine was replaced with an equal amount of cystine during etching, and the final concentration of cystine was 0.1 g / L.

[0138] Comparative Example 2: A method for preparing micropits

[0139] The method for preparing the micro-pits is similar to that in Example 10;

[0140] The difference from Example 10 is that in Comparative Example 2, sodium molybdate was replaced with an equal amount of sodium silicate during etching, and the final concentration of sodium silicate was 0.6 g / L.

[0141] Comparative Example 3: A method for preparing micropits

[0142] The method for preparing the micro-pits is similar to that in Example 5;

[0143] The difference from Example 5 is that Comparative Example 3 uses bismuth ions as heavy ions.

[0144] Comparison of Micropit Parameters in Experiment Example 1

[0145] 1. Experimental sample: The substrate containing micropits prepared in Examples 1-20.

[0146] 2. Experimental method: During the experiment, the density and length of the nuclear track generated on the substrate during irradiation were recorded in real time, and the opening diameter and depth of individual micropits were measured after cleaning and drying.

[0147] 3. Experimental results: The specific experimental results are shown in Tables 1 to 4.

[0148] Table 1. Parameters related to micropits on glass slides as substrates

[0149]

[0150] Table 2. Parameters related to micropits on polyester film substrate.

[0151]

[0152] Table 3. Parameters related to micropits on polycarbonate film substrate

[0153]

[0154] Table 4. Parameters related to micropits on polyimide film substrate

[0155]

[0156] As shown in Tables 1-4 above, this invention obtains nuclear tracks of different densities and lengths by adjusting the heavy ion beam energy and irradiation time. The change in heavy ion beam energy has a significant impact on the density of the nuclear tracks, ranging from 10... 4 ~10 9Furthermore, during the etching process, etching temperature and time directly affect the size and depth of the micropores. As temperature and time increase, the diameter and depth of the micropores gradually increase. However, the addition of an etching inhibitor alters the diameter and depth of the micropores to varying degrees. This demonstrates that by changing the etching temperature and time, micropores of different sizes can be prepared, effectively improving the preparation efficiency and laying the foundation for their application in single-molecule detection.

[0157] Experiment Example 2: Comparison of Micro-pit Shapes

[0158] 1. Experimental samples: Micropits prepared in Example 5 and Example 10, and micropits prepared in Comparative Examples 1-3;

[0159] 2. Experimental method: The sample surface was scanned with a focused electron beam to observe the shape of different micro-pits;

[0160] 3. Experimental Results: Taking Example 5 as an example, the possible effects of controlling heavy ion energy and angle are as follows: By adjusting the bombardment angle, heavy ion energy, etc., a cylindrical hole can be obtained ( Figure 1 A) Conical hole ( Figure 1 B), symmetrical double conical hole ( Figure 1 C), asymmetric double conical hole ( Figure 1 Micropits of shapes such as (D) can be obtained, and unconventional shapes such as hemispherical, ellipsoidal, or teardrop-shaped micropits can also be obtained. These micropits can be further used for single-molecule detection. Example 10 can also obtain micropits of the above shapes (D). Figure 1 Correspondingly, in Comparative Examples 1 and 2, due to the change in the type of corrosion inhibitor, the etching rate control effect decreased during etching, making it impossible to obtain the aforementioned micropit shapes, and over-etching easily occurred, leading to penetration. In Comparative Example 3, the substrate was irradiated with a bismuth ion beam. Since bismuth is a highly inert metallic element, it hardly participates in chemical reactions during ion beam processing, and its material removal relies almost entirely on physical sputtering. Therefore, the etching selectivity was very poor, making precise stop control difficult and unable to control the shape of the micropits.

Claims

1. A method for fabricating micropits for a single-molecule detection chip substrate, characterized in that, Includes the following steps: S1. Irradiation: Heavy ions are accelerated using an accelerator to obtain a heavy ion beam, and then the heavy ion beam is used to bombard the substrate to generate nuclear tracks, thereby obtaining a damaged substrate. S2, Etching: Etching the damaged substrate obtained in step S1 with an etchant to obtain a substrate containing micropits; S3. Cleaning and drying: Clean the substrate containing micro-pits obtained in step S2 with cleaning solution and dry it in a vacuum drying oven to obtain the substrate for single-molecule detection chip. In step S1, heavy ions are accelerated to an energy of 10-1200 MeV using an accelerator. The heavy ions include Br ions and Ar ions. In step S2, the etchant is one of hydrogen fluoride solution, sodium hydroxide solution, potassium hydroxide solution, and fluorosilicic acid solution. The etchant in step S2 also includes a corrosion inhibitor, which is selected from one or more of methionine, histidine, molybdate, and tungstate. In step S2, the etching temperature is 25-80℃, and the etching time is 90s-180min. The diameter of the micropits on the substrate obtained in step S2 is 0.3-45μm, and the depth of the micropits is 0.2-30μm.

2. The method for fabricating micropits for a single-molecule detection chip substrate as described in claim 1, characterized in that, The substrate in step S1 is one of glass, silicon wafer and polymer film; the thickness of the substrate is 0.002~6mm.

3. The method for fabricating micropits for a single-molecule detection chip substrate as described in claim 1, characterized in that, The heavy ion beam described in step S1 is scanned into a rectangular plane using a beam scanner.

4. The method for fabricating micropits for a single-molecule detection chip substrate as described in claim 1, characterized in that, In step S1, during the bombardment of the substrate with accelerated heavy ions, the substrate is bombarded with heavy ions in the atmosphere, and a titanium film is used for vacuum isolation. After bombardment, the density of the nuclear track is 1 × 10⁻⁶ per square centimeter. 4 ~1×10 9 ions.

5. A substrate for a single-molecule detection chip prepared by the preparation method according to any one of claims 1 to 4.

6. A single-molecule detection chip comprising the substrate as described in claim 5.

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